The most dominant means of transmission of HIV and spread of AIDS worldwide is by heterosexual intercourse. Microbicides, compounds that could be used in vaginal and rectal formulations, are increasingly seen as an urgent goal to stop transmission. The protein cyanovirin-N (CV-N) is a highly stable protein that binds to HIV-1 Env gp120 and antagonizes viral entry into host cells and consequent infection. CV-N can be produced recombinantly at large scale, has been found to be effective in blocking HIV-1 transmission in animal studies and is currently in pre-clinical development as a microbicide. Nonetheless, there are potential limitations, including amount of CV-N production required based on measured in vivo efficacy, reliance on a single mode of action and CV-N resistant strains of virus. Limitations of CV-N could be overcome by combining this protein with a second inhibitory agent that binds to a different interaction site in the same molecular target, gp120, and synergizes its antagonist activity with that of CV-N. Recently, we have found that a peptide denoted 12p1 functions as an allosteric dual antagonist of gp120 interactions with host cell receptors and can improve the antiviral activity of CV-N in combination mixtures. We have identified a chemical modification strategy that yields strikingly higher affinity forms of 12p1 for gp120. Further, we have lead data demonstrating that a recombinant chimera of 12p1 and CV-N has enhanced antagonist activity vs CVN alone. The R21 part of this proposal is to examine the usefulness of evolving 12p1-based molecules as possible means to enhance the capability of CV-N as a microbicidal agent. The R33 phase will be to develop promising lead combinations and chimeras, including advanced agent design based on structural mechanism of action; to define in vitro and in vivo efficacy of lead chimeras and combinations and to examine synergies with other agents in the microbicide field; and to develop production approaches for the protein and peptide agents derived in this work. [unreadable] [unreadable] We will prioritize two specific aims in the R21 project. [1] Produce recombinant CVN-12p1 chimeras with varying linkers and determine efficacy of chimera candidates versus that of the separate CV-N and 12p1 components. Work on this aim will identify CVN-12p1 chimeras with optimized HIV-1 Env gp120 antagonism and potential as recombinant AIDS microbicide candidates. [2] Generate and screen affinity-enhanced conjugate 12p1 variants for non-covalent combination with CVN and identify optimized mixture candidates. Work in this aim will provide alternative microbicide candidates that combine recombinant CV-N with synthetic 12p1 conjugates. [unreadable] [unreadable] The major milestone at the end of the R21 phase will be identification of 12p1/CVN combinations, both chimeras and mixtures, which will have antagonist activities that are enhanced vs CV-N alone and which hence will be recommended for development as microbicides in their own right. The CVN-12p1 chimeras and noncovalent combinations will introduce a microbicide with a novel mechanism of action, substantially overcome potency and resistance limitations of CV-N alone and hence lead to improved microbicide candidates to combat global AIDS transmission. [unreadable] [unreadable] [unreadable]